PROJECT SUMMARY Gammaherpesviruses (?HVs) are lifelong infections that are critically regulated by host immunity, with immune suppressed individuals at a significantly increased risk for ?HV-associated diseases including malignancies. There remain limited therapeutic options to treat ?HV-associated disease, a problem exacerbated by ?HV latency, a quiescent state of infection, characterized by limited gene expression and low immunogenicity. Latent infection is further problematic in that latently infected cells have the capacity to reactivate, resulting in de novo virus replication and infectious virus particles. The challenge of viral quiescence and re-emergence has been increasingly identified in other contexts, including HIV-infected individuals on highly active antiretroviral therapy (HAART). In both of these contexts, there have been efforts to specifically target quiescent viral reservoirs, either to induce a permanent, reactivation-incompetent state or to specifically induce reactivation in these quiescent cells, combined with strategies to disrupt virus replication. If successful, these approaches could have profound effects, ranging from new therapies to disrupt ?HV latency-associated cancers to achieving an HIV cure. One major challenge in this line of investigation is that we still have an incomplete understanding of the factors that influence the reactivation-competency of viral latency. Whether for the express purpose of altering ?HV latency and reactivation during chronic infection, or as a byproduct of treatments for HIV, transplant or malignancies, effective monitoring of ?HV latency and reactivation at the single-cell level is a relevant and timely goal. This proposal is based on the following: i) ?HV infections are ubiquitous chronic infections in homeostatic balance with host immunity, and ii) latency and reactivation-competency will be altered by regimens currently pursued for HIV cure and for tumor immunotherapy. It is important to note that whether these alterations would be beneficial or detrimental to ?HV control remains unknown. This R21 proposal seeks to develop new methods to define the spectrum of reactivation-competency in ?HV infected cells at the single-cell level. By integrating this method with an analysis of clinically-relevant methods to induce viral reservoirs, these studies will permit monitoring of ?HV status across a variety of interventions (e.g. from HIV cure therapies to immunotherapy) in unfractionated samples (e.g. peripheral blood), to provide a single, quantitative and clinically tractable platform. We propose to develop this single-cell analysis method in the ?HV68 model (SA1) and the human ?HVs, EBV and KSHV (SA2). Notably, these studies focus on reactivation-inducing stimuli that are actively being investigated to induce reactivation in either the ?HVs or HIV. In total, this R21 proposal seeks to develop new single-cell methods to characterize ?HV latently infected cells and their reactivation-competence, to gain clinically-relevant insights that will ultimately inform efforts to directly target quiescent viral reservoirs.